Durand R, Job C, Zarling D A, Teissère M, Jovin T M, Job D
EMBO J. 1983;2(10):1707-14. doi: 10.1002/j.1460-2075.1983.tb01646.x.
The template properties of left-handed synthetic polymers, the Z* form of poly[d(G-C)] and the Z form of poly[d(G-m5C)], have been investigated using an eucaryotic RNA polymerase, the class II enzyme from wheat germ. Results from a comparative kinetic study of transcription using the polynucleotide substrates in the B and Z conformations are reported. Optimal conditions for enzyme activity compatible with the preservation of the desired template conformation were determined. On the basis of several criteria, both physical (c.d. spectra of the polymers, sedimentability of the Z* form) and biochemical, it was demonstrated that the left-handed conformations of poly[d(G-C)] and poly[d(G-m5C)] serve as templates for wheat germ RNA polymerase II. The level of incorporation was less than that exhibited by the B form of poly[d(G-C)], the relative activity being a function of the precise experimental conditions. Activity ratios (Z*/B or Z/B) ranged from 0.1 to 0.5. The effect of various incubation parameters, including pH, salt concentration, temperature, and the presence of dinucleoside monophosphate primers were investigated. The Km values for nucleoside triphosphate substrates were slightly smaller for the Z* form of poly[d(G-C)] than for the B conformation. Titration of DNA (Z* or B) with enzyme and reciprocal experiments suggested that the reduced activity of left-handed templates might derive from the availability of fewer and/or lower affinity sites for initiation and/or translocation on these templates. Specific antibodies raised against left-handed DNA strongly inhibited the observed transcription of Z* and Z DNAs by wheat germ RNA polymerase II.
利用真核生物RNA聚合酶——来自小麦胚芽的II类酶，研究了左手性合成聚合物、聚[d(G-C)]的Z形式和聚[d(G-m5C)]的Z形式的模板特性。报道了使用处于B构象和Z构象的多核苷酸底物进行转录的比较动力学研究结果。确定了与所需模板构象保存相容的酶活性最佳条件。基于几个标准，包括物理标准（聚合物的圆二色谱、Z形式的沉降性）和生化标准，证明了聚[d(G-C)]和聚[d(G-m5C)]的左手性构象可作为小麦胚芽RNA聚合酶II的模板。掺入水平低于聚[d(G-C)]的B形式所表现出的水平，相对活性是精确实验条件的函数。活性比（Z*/B或Z/B）范围为0.1至0.5。研究了各种孵育参数的影响，包括pH、盐浓度、温度以及二核苷单磷酸引物的存在。聚[d(G-C)]的Z形式的核苷三磷酸底物的Km值比B构象的略小。用酶对DNA（Z或B）进行滴定以及反向实验表明，左手性模板活性降低可能源于这些模板上起始和/或易位的位点较少和/或亲和力较低。针对左手性DNA产生的特异性抗体强烈抑制了小麦胚芽RNA聚合酶II对Z*和Z DNA的转录。
